Comparison of two‐photon excitation laser scanning microscopy with UV‐confocal laser scanning microscopy in three‐dimensional calcium imaging using the fluorescence indicator Indo‐1

Abstract

SummaryTwo-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6-17, which enhances the contrast by a factor of 6-21; (2) a lower rate of photobleaching by a factor of 2-4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0 . 18-mW laser power with a 2 . 6-f pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0 . 09-mW laser power with a 4 . 9-f pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.

Confocal laser scanning microscopy of calcium dynamics in living cells

“…For example, in the absence of antioxidants, UV irradiation can eventually convert reactive forms of indo-1 into Ca 2ϩ -insensitive species, and this conversion cannot be compensated for by ratiometric calibrations (Scheenen et al, 1996). Moreover, compared to laser illumination in the visible light spectrum, UV illumination can generate greater phototoxicity in some cells tested (Sako et al, 1997).…”

Calcium dynamics in single spines during coincident pre- and postsynaptic activity depend on relative timing of back-propagating action potentials and subthreshold excitatory postsynaptic potentials

“…In line scan imaging both scan directions and external detection through the condenser were used for signal collection. This allowed us to use an average laser power (Յ4-9 mW in the focal plane, depending on depth below slice surface) that was below the threshold (9-12 mW, measured in the system used) for photodamage (15). Transmission and epifluorescence signals were recorded by photomultiplier tubes (R6357, Hamamatsu Photonics, Herrsching, Germany) and summed off-line.…”

Measurement of Intracellular Calcium

“…The mechanisms responsible for this high resolution are, however, quite different from that of CLSM. Because the probability of the simultaneous excitation by two individual photons is proportional to the square of the photon concentration, fluorophores far from the plane of focus cannot be excited with enough power with TPLSM, resulting in high spatial resolution (334). In the case of the CLSM, not only the focal plane but also the areas surrounding the plane of focus are excited, but the pinhole blocks emission light from the out-of-focus plane from reaching the detectors.…”